Ikaros is the founding member of a small family of C2H2 zinc finger DNA-binding proteins and has been found to play critical roles in lymphocyte development and tumor suppression. Like many of the approximately 800 C2H2 zinc finger proteins encoded by mammalian genomes, Ikaros contains multiple tandem zinc fingers within its DNA-binding domain. Since the discovery of Ikaros in 1992, much has been learned about its biological functions from elegant studies of Ikaros mutant mice. However, its precise intracellular functions and mechanisms of action have remained elusive, largely because it does not appear to function as a typical transcription factor and because target genes responsible for its most important biological functions have been difficult to identify. We have come to realize that the only way we can fully understand its mechanisms of action, and the only way we can evaluate hypotheses that have emerged from our biochemical studies, is to study Ikaros domains and biochemical activities in a native physiological setting. Toward this end, we have introduced specific mutations into the endogenous Ikzf1 locus, which encodes Ikaros. Two mutant strains have been characterized to date. These two strains contain deletions of exons encoding the first and last zinc fingers of the DNA-binding domain. These mutant mice were created (1) to test a hypothesis that these two fingers regulate binding to distinct sets of target genes, (2) to facilitate the discovery of new Ikaros target genes, and (3) to contribute to the broader C2H2 zinc finger field by exploring the biological reason for the existence of tandem arrays of zinc fingers. Importantly, we have found that each mutant strain exhibits a select subset of the phenotypes previously described in Ikaros null mice, providing strong evidence that the two fingers regulate different target genes and biological functions. In Aim 1, we will further characterize selective phenotypes of the mutant strains to better understand how these two fingers differentially regulate previously described target genes. In Aim 2, an unbiased approach that takes advantage of the selective phenotypes of the mutant strains will be used to identify and characterize new Ikaros target genes. Finally, we will characterize a third mutant mouse strains that lacks a key residue involved in Ikaros multimerization, and we will begin to use a similar approach to examine the functional significance of two co-repressors that are known to interact with Ikaros. PUBLIC HEALTH RELEVANCE: Ikaros proteins are critical regulators of many stages of human hematopoiesis, yet their precise functions and mechanisms of action remain poorly understood. Furthermore, the IKZF1 gene, which encodes human Ikaros, is deleted or partially deleted in over 80% of patients with BCR- ABL+ acute lymphoblastic leukemia (ALL) and over 20% of high-risk pediatric patients with B-cell progenitor ALL. A better understanding of the functions and mechanisms of action of Ikaros will enhance our understanding of normal hematopoiesis and may lead to novel therapeutic strategies for leukemia patients. PUBLIC HEALTH RELEVANCE: Ikaros family proteins are critical regulators of many stages of human hematopoiesis, yet their precise functions and mechanisms of action remain poorly understood. Furthermore, the IKZF1 gene, which encodes human Ikaros, is deleted or partially deleted in a high percentage of patients with specific classes of acute lymphoblastic leukemia. A better understanding of the functions and mechanisms of action of Ikaros will enhance our understanding of normal hematopoiesis and may lead to novel therapeutic strategies for leukemia patients.